Understanding the factors that control the fidelity of DNA polymerases is a problem of great fundamental and practical importance. Here we propose to exploit our recent advances and to continue the use of computer simulation approaches to gain a deeper insight that will complement the experimental progress in projects 1 and 3. It is proposed to use reliable theoretical tools for structure/fidelity correlation and to refine this correlation by a constant feedback from kinetic, binding, and structural experiments. The proposed projects include: (i) Establishing the cleavage mechanism of the PaO bond of dNTP substrates in the polymerase active site by a concerted use of different theoretical approaches, ranging from ab initio QM-MM free energy calculations to constraint DFT free energy calculations and to systematic empirical valence bond studies, (ii) Refining our calculations of substrate binding free energies as well as calculations of the effects of pol pi mutations on the these binding energies, (iii) Exploring factors that control the fidelity of polymerases while| paying special attention to the allosteric transfer of information between the base binding site and the! chemical reaction site. The allosteric information transfer will be studied by calculating the coupling between different residues and the transition state (TS) using the correlation matrix formalism. (iv)Exploring the nature of the TS for incorporating right (R) and wrong (W) nucleotides, including the study of the effect of the protein conformational landscape on the nature of the TS. These studies will be helped by structural studies of transition state analogues (TSAs) with correct and incorrect base pairs, (v) Determining the structure and binding energy of different TSAs that will be examined experimentally by our coworkers (Project 3) and explored theoretically. This study will teach us about the relationship between the TSAs and the actual TS and will help in verifying the calculated TS properties. All the above studies will involve constant feedback from the experimental counterparts of the project. This collaboration will help us develop a more general and coherent view about the nature of DNA polymerase fidelity.